Various food products, e.g. soups, sauces and seasonings, comprises flavoring agents obtained by hydrolysis of proteinaceous materials. Conventionally this hydrolysis is brought about using strong hydrochloric acid, followed by neutralization with sodium hydroxide. However, such chemical hydrolysis leads to severe degradation of the amino acids obtained during the hydrolysis, and also to hazardous byproducts formed in the course of this chemical reaction. Therefore increasing concern over the use of flavoring agents obtained by chemical hydrolysis has lead to the development of enzymatic hydrolysis processes.
Enzymatic hydrolysis processes aim at obtaining a high degree of hydrolysis (DH), and this is usually attained using a complex of unspecific acting proteolytic enzymes (i.e. unspecific acting endo- and exo-peptidases). In this way e.g. WO 94/25580 describes a method for hydrolyzing proteins by use of an unspecific acting enzyme preparation obtained from Aspergillus oryzae. Specific acting proteolytic enzymes have not been used for this purpose, because such enzymes only lead to an inadequate degree of hydrolysis.
Only relatively few specific acting proteolytic enzymes have been reported. Thus proteolytic enzymes preferentially cleaving peptides at glutamoyl-peptide bonds (Glu-specific proteases) obtained from Staphylococcus aureus [Drapeau G R, Boily Y and Houmard J; J. Biol. Chem. 1972 247 (20) 6720-6726], from Actinomyces sp. [Mosolova O V, Rudenskaya G N, Stepanov V M, Khodova O M and Tsaplina I A; Biokhimiya 1987 52 (3) 414-422], from Streptomyces griseus [Yoshida N, Tsuruyama S, Nagata K, Hirayama K, Noda K and Makisumi S; J. Biochem. 1988 104 451-456], from Streptomyces thermovulgaris [Khaldarova N Y, Rudenskaya G N, Revina L P, Stephanov V M and Egorov N S; Biochimiia Moscow Eng. 1989 54 (1) 32-38], from Bacillus subtilis [Niidome T, Yoshida N, Ogata F, Ito A and Noda K; J. Biochem. 1990 108 965-970], and from Bacillus licheniformis [WO 91/13554 A1], have been reported.
Glu-specific proteases primarily find applications in studies of protein structures. However, use of a Glu-specific Staphylococcus aureus protease for modifying the solubility and structural properties of casein has been reported [Chobert J-M, Sitohy M Z and Whitaker J R; J. Agric. Food Chem. 1988 36 220-224], and WO 91/13554 A1 describes the use of the Bacillus licheniformis for obtaining a limited specific hydrolysis of proteins.
Whereas glutamine (Gin) is almost tasteless, glutamic acid (Glu), whether free or peptide bound, plays an important role for the flavor and palatability of protein hydrolysates. Glutamic acid may be formed by converting free glutamine to glutamic acid. This conversion (i.e. deamidation) is inherently taking place during conventional chemical hydrolysis using strong hydrochloric acid.
Alternatively glutamic acid may be formed from free glutamine by the action of a glutaminase (L-Glutaminase, EC 3.5.1.22 or D-Glutaminase, EC 3.5.1.35). However, substantial amounts of the glutamine may be lost as it spontaneously converts to pyroglutamine.
In contrast to glutaminase, peptidoglutaminase (PGases) acts on peptide bound glutamine. Two types of peptidoglutaminases are known. Peptidyl-glutaminase (EC 3.5.1.43; Peptidoglutaminase I) specific for glutamine substituted at the .alpha.-amino group, and Protein-glutamine glutaminase (EC 3.5.1.44; Peptidoglutaminase II) specific for glutamine substituted at the carboxyl position or both the .alpha.-amino and carboxyl positions. Peptidoglutaminases obtained from Aspergillus japonicus, Bacillus circulans, Cryptococcus albidus, and Debaryomyces kloecheri have been reported [Kikuchi M & Sakaguchi K; Agr. Biol. Chem. 1973 37 (4) 719-724].
Peptidoglutaminases are known and useful for modifying food proteins. Structural modifications of proteins improve their functional properties and extends their use as food ingredients. Thus U.S. Pat. No. 5,082,672 describes a method of treating food proteins by deamidation with a peptidoglutaminase, by which method the solubility, emulsification, foaming and other functional properties of soy proteins become improved. However, U.S. Pat. No. 5,082,672 also report that the ability of peptidoglutaminase to deamidate intact soy protein is limited due to its large molecular size and/or unique conformation. Therefore U.S. Pat. No. 5,082,672 teach initial degradation of the substrate by hydrolysis with Alcalase.TM., an un-specific acting endo/exo-peptidase complex obtained from Bacillus licheniformis, and/or heat treatment, and find that pre- and post-heat treatment of hydrolyzed soy protein produced the greatest degree of deamidation.
U.S. Pat. No. 3,857,967 describes a process for preparing food and beverages with a peptidoglutaminase obtained from Bacillus circulans. Also, in order to obtain the greatest degree of deamidation, U.S. Pat. No. 3,857,967 teach initial degradation of the proteinaceous substrate by use of un-specific acting endo/exo-peptidases.
Mimouni et al. [Mimouni B, Raymond J, Merle-Desnoyers A M, Azanza J L & Ducastaing A; Journal of Cereal Science 1994 21 153-165] describe a combined acid deamidation and enzymatic hydrolysis for improvement of the functional properties of wheat gluten. More particularly, Mimouni et al. describe acid deamidation combined with the use of unspecific acting endo-peptidases.